Combination HIV therapeutic

ABSTRACT

Embodiments of the present invention are directed to particles having a Bryoid and a HDAC inhibitor for the treatment of latent viral disease.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15,576,190 filed Nov.21, 2017, which claims priority to U.S. Provisional Application Ser. No.62/165,444 filed May 22, 2015, which is incorporated by reference in itsentirety herein.

STATEMENT REGARDING FEDERAL SPONSORSHIP

Embodiments of the present invention were not conceived nor reduced topractice with Federal funds or sponsorship.

FIELD OF THE INVENTION

Embodiments of the present invention relate to the field of latent viraldiseases and articles of manufacture, compositions and methods for thetreatment of such diseases.

BACKGROUND OF THE INVENTION

Antiretrovirus therapy (ART) is an indispensable life-saving therapy formillions of HIV+ individuals. However, the persistence of latentHIV-infected cellular reservoirs remains the last major hurdle to viruseradication. Latently infected cells represent a permanent source ofpotential viral reactivation. For this reason, the eradication of viralreservoirs is now the major goal for HIV-1 therapeutics (Richman et al.,2009).

SUMMARY OF THE INVENTION

Embodiments of the present invention are directed to medicaments,methods of treatment and articles of manufacture in the form of aparticle for treating latent viral disease. As used herein, the term“medicament” broadly means any agent used in the treatment of a disease,such as, for example, without limitation, tablets, capsules, gelcaps,powders, patches, emulsion, suspensions and solutions which areadministered orally, rectally, buccally, sublingually, subcutaneously,intramuscularly, intravenously and intraperitoneal.

One embodiment directed to a medicament comprises a Histone Deacetylase(HDAC) inhibitor, and a Bryoid, for treating a latent viral disease. Oneembodiment features a Bryoid is selected from the group of Bryostatinsconsisting of Bryostatin 1-20 and other known or identified Bryostatins.One embodiment features a HDAC inhibitor is selected from the groupconsisting of valproic acid, Vorinostat, Romidepsin and Panobinostat.

Embodiments of the present invention feature the administration of aBryoid in a dose effective with the dose of the HDAC inhibitor. TheBryoid is administered in an effective dose range of 10 to 100microgram/Kg subject every other day for up to 180 days.

Embodiments of the present invention feature the administration of HDACinhibitor in a dose effective with the Bryoid. The HDAC inhibitor isadministered in an effective dose range of 10 to 100 mg/Kg subject everyother day for up to 180 days.

One embodiment of the invention features the HDAC inhibitor and Bryoidcarried by one or more particles. As used herein, the term “carried by”refers to any configuration in which the HDAC inhibitor and Bryoid areassociated with the particle. The term encompasses by way of examplewithout limitation one or more of the HDAC inhibitor and Bryoiddistributed throughout the particle, or on the surface of the particleor in a section of the particle.

One embodiment features one or more particles, in which the particle hasa core, at least one surrounding material and an outer surface. The corehas a mixture of a hydrophilic material and an HDAC inhibitor. Thesurrounding material has a mixture of a hydrophobic material and aBryoid. The surrounding material envelopes the core and the outersurface surrounding the surrounding material. As used herein, the term“mixture” denotes a distribution whether in solution, in suspension oras an emulsion.

One embodiment of the invention features one or more particles fortreating a latent viral disease having an outer surface. The virusassociated with the latent viral disease has one or more viralcomponents. The one or more particles comprise one or more ligandsspecific for the viral component and the one or more ligands associatedwith the outer surface of the one or more particles. For example,without limitation, the viral components comprise protein markersspecific for Human Immunodeficiency Virus (HIV) and the particle surfacecomprises ligand such as antibodies, aptamers and similar constructs.

One embodiment of the invention features one or more particles whichhave one or more upregulating ligands to upregulate CD-4 cells. The oneor more upregulating ligands are associated with the surface.

One embodiment directed to a method of treating a latent viral disease,comprises the step of administering an effective amount of a HistoneDeacetylase (HDAC) inhibitor and an effective amount of a Bryoid. Oneembodiment of the method features a Bryoid selected from the group ofBryostatins consisting of Bryostatin 1-20 and other known or identifiedBryostatins. One embodiment of the method features a HDAC inhibitor isselected from the group consisting of valproic acid, Vorinostat,Romidepsin and Panobinostat.

In one aspect of the method, the method Bryoid is administered in aneffective dose range of 10 to 50 microgram/Kg subject. In one aspect ofthe method, the HDAC inhibitor is administered in an effective doserange of 10 to 100 mg/Kg subject.

One embodiment of the invention features the HDAC inhibitor and Bryoidcarried by one or more particles. For example, without limitation oneembodiment features a method wherein the one or more particles has acore, at least one surrounding material and an outer surface. The corehas a mixture of a hydrophilic material and the HDAC inhibitor, and thesurrounding material has a mixture of a hydrophobic material and theBryoid. The surrounding material envelopes the core and the outersurface surrounds the surrounding material.

One embodiment of the method features the one or more particles havingan outer surface and the virus associated with the latent viral diseasehaving one or more viral components. The one or more particles compriseone or more ligands specific for the viral component, with the one ormore ligands associated with the outer surface of the one or moreparticles.

One embodiment of the method features the one or more particlescomprising one or more upregulating ligands to upregulate CD-4 cells.The one or more upregulating ligands are associated with the surface.

A further embodiment of the present invention is directed to an articleof manufacture comprising a particle. The particle has a core, at leastone surrounding material and an outer surface. The core has a mixture ofa hydrophilic material and a Histone Deacetylase (HDAC) inhibitor. Thesurrounding material has a mixture of hydrophobic material and a Bryoid.The surrounding material envelopes the core and the outer surfacesurrounds the surrounding material. The particle is for treating alatent viral disease.

One embodiment of the invention features a particle wherein the virusassociated with the latent viral disease has one or more viralcomponents. The particle comprises one or more ligands specific for theviral component. The one or more ligands are associated with the outersurface of the particle.

One embodiment of the invention features a particle further comprisingone or more upregulating ligands to upregulate CD-4 cells. The one ormore upregulating ligands are associated with the outer surface.

One embodiment of the invention features a particle wherein the Bryoidis selected from the group of Bryostatins consisting of Bryostatin 1-20and other known or identified Bryostatins. One embodiment features aparticle wherein the HDAC inhibitor is selected from the groupconsisting of valproic acid, Vorinostat, Romidepsin and Panobinostat.

These and other features and advantages will be apparent to thoseskilled in the art upon viewing the drawings which are described inbrief below and studying the Detailed Description of the Invention whichfollows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a cross sectional view of a particle embodying featuresof the present invention; and

FIG. 2 depicts a schematic view of an apparatus for making the particleof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Different investigators have suggested that reactivation of the latentreservoirs with immunoactivation therapy would allow effective targetingand possible eradication of the virus. It is thought that viralreactivation by this therapy would result in lytic cell death of CD4+ Tcells because of the cytopathic effect of the virus or throughrecognition of infected cells by the immune system. In addition, viralreactivation in the presence of ART would prevent new infections. Inthis sense a Histone Deacetylase (HDAC) inhibitor, Vorinostat, induced asignificant and sustained increase in HIV transcription from latency insome HIV-infected patients but failed to clear HIV-1 reservoirs. Theseresults indicate that additional strategies will be needed to eliminatelatently infected cells.

Embodiments of the present invention feature Protein Kinase C (PKC)agonists such as the non-tumorigenic Bryoids combined with HDACinhibitors to purge latent HIV-1 from cellular reservoirs. Currently,over 22 million people have died from AIDS and there are over 42 millionpeople living with HIV/AIDS worldwide. In the United States, anestimated 1 million people are currently living with HIV andapproximately 40,000 infections occur each year. There is no vaccineagainst HIV and AIDS, if untreated, will lead to the death of over 95%of infected individuals 10 years post-infection. HIV infects severalcell types during the course of infection and progression to acquiredimmune deficiency syndrome (AIDS).

The persistence of latent HIV-infected cellular reservoirs representsthe major hurdle to virus eradication with anti-retroviral therapy(ART), since latently infected cells remain a permanent source of viralreactivation. It has been hypothesized that intensification of ART couldreduce the residual viremia but recent studies strongly suggest thatthis is not the likely scenario.

Moreover, ART is problematic because of long-term toxicity, inhibitorresistance, and the inability to target persistent reservoirs.Therefore, other pharmacological approaches targeting the HIV-1reservoir have been suggested by several investigators as a promisingstrategy to develop new drugs able to activate latent HIV-1 withoutinducing a global T cell-activation.

HIV-1 infects several cell types during the course of infection andprogression to AIDS. In the absence of ART, HIV-1 replication is activein most of the infected cells and in the majority of patients. However,HIV-1 establishes long-term infection in a small pool of memory CD4+ Tcells and in other cell types, which contain integrated buttranscriptionally silent HIV provirus. These latently infected cellsconstitute a viral reservoir in which a replication-competent form ofthe virus persists with more stable kinetics than the main pool ofactively replicating virus.

Although ART is undoubtedly a life-saving therapy for millions of AIDSpatients, the persistence of latent HIV-infected cellular reservoirsrepresents the major hurdle to virus eradication, since latentlyinfected cells remain a permanent source of viral reactivation. As aresult, a sudden rebound of the viral load after interruption of HAARTis generally observed. For this reason, eradication of viral reservoirsis at present the major goal for HIV-1 therapeutics.

Early introduction and intensification of ART have been suggested todiminish the frequency of latently infected memory CD4+ T cells.However, a recent report has shown that ART intensification does notreduce residual viremia in a small cohort of patients. Moreover, it isbelieved that even a few, or a single, residually infected cell would besufficient to produce systemic viremia upon ART interruption. Therefore,it has been hypothesized that reactivation of the latent reservoirscould allow effective targeting and possible eradication of the virus.

It is thought that viral reactivation would result in lytic cell deathof CD4+ T cells because of the cytophatic effect of the virus or throughrecognition of infected cells by the immune system. In addition, viralreactivation in the presence of ART would also prevent new infectionevents. Developing drugs directed against different targets of the HIVcycle is urgently needed, especially the development of drugs able todiminish or eradicate latent reservoirs. This therapy should not inducepolyclonal T cell activation.

The present invention features Protein Kinase C (PKC) agonists such asthe non-tumorigenic Bryoids combined with Histone Deacetylase (HDAC)inhibitors to purge latent HIV-1 from cellular reservoirs. HDAC is anenzyme that removes acetyl groups from DNA bound histone proteins,affecting gene expression and contributing to HIV latency. Inhibitors ofHDAC have been shown to reverse latency in vitro, ex vivo, and recentlyin a human clinical trial. Vorinostat, a HDAC inhibitor, failed toeliminate HIV-1 reservoirs in patients. Bryoids such as Bryostatin-1, aswell as many PKC agonists, activates cellular transcription factors suchas NF-κB that binds the HIV-1 promoter and regulates its transcriptionalactivity. In HIV-1 latency the viral promoter is less accessible tocellular transcription factors because nuclear histones surrounding theviral promoter are deacetylated (compacted chromatin). Thus, HDACinhibitors may increase the acetylation of histones (relaxed chromatin)and then transcription factors may have an easier access to the HIVpromoter.

One embodiment of the present invention features the administration of aBryoid in a dose effective with the dose of the HDAC inhibitor. As usedherein, the term “administer” or “administration” refers to the takingor receiving of a medicament in an effective manner, such as takingorally a tablet, capsule, powder, gelcap, liquid, suspension, emulsionor the like orally; or a liquid, emulsion or suspension for injection.The Bryoid is administered in an effective dose of 10 to 100microgram/Kg subject every other day for up to 180 days.

One embodiment of the present invention features the administration ofHDAC inhibitor in a dose effective with the Bryoid. The HDAC inhibitoris administered in an effective dose of 10 to 100 mg/Kg subject everyother day for up to 180 days.

One embodiment of the invention features the HDAC inhibitor and Bryoidcarried by one or more particles. Turning now to FIG. 1, a particlehaving features of the present invention, generally designated by thenumeral 11, is depicted. The particle has a core 15, at least onesurrounding material 17 and an outer surface 21. The core 15 has amixture of a hydrophilic material and an HDAC inhibitor. The surroundingmaterial 17 has a mixture of a hydrophobic material and a Bryoid. Thesurrounding material envelopes 17 the core 15 and the outer surface 21surrounding the surrounding material 17.

The core 15 is an aqueous solution that forms a mixture with the HDACinhibitor. The aqueous solution may comprise other constituents such assalts and buffering agents.

The surrounding material 17 is selected from hydrophobic compositionsincluding phospholipids and like materials which form substantiallyuniform mixtures with a selected Bryoid. For example, withoutlimitation, the phospholipid is selected from one or more of the groupconsisting of phosphatidylcholine (PC), phosphatidylglycerol (PG),phosphatidylserine (PS), dimyristoylphosphatidylcholine (DMPC),dimyristoylphosphatidylglycerol (DMPG), phosphatidylethanolamine (PE),and polyethylene glycol conjugated distearylphosphatidylethanolamine(either DSPE-PEG₂₀₀₀ or DSPE-PEG₃₅₀₀). Hydrophobic compositions includeby way of example, without limitation α-tocopherol (vitamin E) andcholesterol. The phospholipids forming the hydrophobic material aredepicted as a hydrophilic head 31 and a hydrophobic tail 33.

The virus associated with the latent viral disease has one or more viralcomponents. For example, without limitation, the viral componentscomprise protein markers specific for Human Immunodeficiency Virus(HIV). As depicted, the outer surface 21 of the particle 11 comprisesone or ligands 23 such as antibodies, nanobody, dual-variable domainligands and similar constructs which bind to such protein markers. Theantibody depicted is a broadly neutralizing antibody (bNAb).

As depicted, the particle has one or more upregulating ligands toupregulate CD-4 cells, an anti-PD-L1 antibody designated by the numeral25. The one or more upregulating ligands are associated with thesurface, similar to the ligand to the protein markers. That is, the headgroups 31 of the phospholipids are modified to covalently carry aligand.

As depicted, one or more head groups of one or more phospholipidcompositions carry a polyethylene glycol modification 35. Polyethyleneglycol modification of the phospholipid conveys decreased recognition byphagocytes.

Embodiments of the present invention feature targeting a combination ofa Bryoid and an HDAC inhibitor co-encapsulated in a long-circulationpegylated immunonanosomes with coatings of broadly neutralizingantibodies and anti-PD-L1 nanobodies, as shown in FIG. 1, will provideefficient HIV latency activation and immunological depletion of latentreservoirs while significantly reducing systemic toxicities of bothBryostatin-1 and the HDAC inhibitor.

Using an in vitro model of HIV-1 latency, Jurkat-LAT-GFP, Bryostatin-1re-activates HIV-1 latency in T cells via classical PKCs pathways.Bryostatin-1, at concentrations higher than 10 nM, induced translocationof cPKCs to the plasma membrane, and activated the canonical NF-κB andMAPKs (JNK and ERK) pathways.

In contrast, lower concentrations of Bryostatin-1 (10 nM) translocatedcPKCs and Ras-GRP1 to the endoplasmic reticulum, activated ERK and thenuclear phosphorylation of p65 that fully reactivates HIV-1 latency. Lowconcentrations of Bryostatin-1 also down-regulated the expression of thehuman HIV-1 receptors CD4 and CXCR4 and prevent de novo HIV-1 infection(Perez, et al., 2010). Low concentrations of Bryostatin-1 activate thecPKC-Ras-Raf-ERK pathway and synergize with an HDAC inhibitor, vaiproicacid (VPA), to activate the transcription factor SP1.

Transcriptome studies found that low vs. high concentrations ofBryostatin-1 at 10 and 100 nM differentially regulate gene expression inT cells. Therefore, therapeutic activity can be achieved atconcentrations that do not activate signal transduction pathways thatmay result in negative side effects.

Bryostatin-1 antagonized HIV-1 latency ex vivo in PBMC isolated fromHIV-1 patients, and Bryostatin-1 at the doses of 10 and 20 μg/m2 did notinduced significant adverse events in HIV-1 patients in a Phase Iclinical study, Madrid, Spain (ClinicalTrials.gov NCT02269605).

In vitro studies suggest that very low concentrations of Bryostatin-1(1-10 nM) synergizes with HDAC inhibitors such as valproic acid toantagonize HIV-1 latency (Perez et al., 2010). Thus, the therapeuticactivity of Bryostatin-1 can be drastically improved in humans byutilizing a HDAC inhibitor. Our research indicates that combinationtherapy will be most effective, and reduce the therapeutic concentrationof a Bryoid from 10 nM to 1 nM reducing systemic toxicities. Toxicitieswill be further reduced by encapsulating the combination therapeutic inliposomes which have been clinically shown to significantly reduce thein vivo toxicity of therapeutic drugs, e.g. the anti-fungal,amphotericin B.

The particle 11, as described, nanoencapsulates a non-tumorogenic Bryoidsuch as Bryostatin-1, which is quite hydrophobic in the lipid bilayer ofa phospholipid nanosomes that are small, uniform liposomes, andco-encapsulate an HDAC inhibitor such as Romidepsin or Panobinostat inthe aqueous core. Particles, of the type described in FIG. 1, are madein a process for the formation of small, uniform liposomes as describedin U.S. Pat. No. 8,637,074 to Castor (2014).

Bryostatin-1 is encapsulated at concentrations of 1 to 100 nm with apreference of 1 to 10 nM and an HDAC inhibitor at concentrations of 30to 1,000 nM with a preference of 30 to 100 nM. The utility of theco-encapsulation is that both drugs will reach their intended target atthe same time, will be guided to the target with broadly neutralizingantibodies and the anti-PD-L1 nanobodies will keep CD4+ T-cellsactivated for clearing the activated HIV-1 virus. The immunonanosomeswill further reduce systemic toxicities while pegylation will increaseresidence time of the circulating nanoparticle increasing therapeuticefficacy and overall therapeutic index.

Targeting a combination of a Bryoid and an HDAC inhibitorco-encapsulated in a long-circulation pegylated immunonanosomes withcoatings of broadly neutralizing antibodies and anti-PD-L1 nanobodies,as shown in FIG. 1, will provide efficient HIV latency activation andimmunological depletion of latent reservoirs while significantlyreducing systemic toxicities of both Bryostatin-1 and the HDACinhibitor.

Using an in vitro model of HIV-1 latency, Jurkat-LAT-GFP, we have shownthat Bryostatin-1 re-activates HIV-1 latency in T cells via classicalPKCs pathways. Bryostatin-1, at concentrations higher than 10 nM,induced translocation of cPKCs to the plasma membrane, and activated thecanonical NF-κB and MAPKs (JNK and ERK) pathways.

In contrast, lower concentrations of Bryostatin-1 (10 nM) translocatedcPKCs and Ras-GRP1 to the endoplasmic reticulum, activated ERK and thenuclear phosphorylation of p65 that fully reactivates HIV-1 latency. Lowconcentrations of Bryostatin-1 also down-regulated the expression of thehuman HIV-1 receptors CD4 and CXCR4 and prevent de novo HIV-1 infection(Perez, et al., 2010). We also found that low concentrations ofBryostatin-1 activate the cPKC-Ras-Raf-ERK pathway and synergize with anHDAC inhibitor, valproic acid (VPA), to activate the transcriptionfactor SP1.

Transcriptome studies found that low vs. high concentrations ofBryostatin-1 at 10 and 100 nM differentially regulate gene expression inT cells. Therefore, therapeutic activity can be achieved atconcentrations that do not activate signal transduction pathways thatmay result in negative side effects.

Bryostatin-1 antagonized HIV-1 latency ex vivo in PBMC isolated fromHIV-1 patients, and Bryostatin-1 at the doses of 10 and 20 μg/m2 did notinduced significant adverse events in HIV-1 patients in a Phase Iclinical study, Madrid, Spain (ClinicalTrials.gov NCT02269605).

In vitro studies that very low concentrations of Bryostatin-1 (1-10 nM)synergizes with HDAC inhibitors such as valproic acid to antagonizeHIV-1 latency (Perez et al., 2010). Thus, the therapeutic activity ofBryostatin-1 can be drastically improved in humans by utilizing a HDACinhibitor. Our research indicates that combination therapy will be mosteffective, and reduce the therapeutic concentration of a Bryoid from 10nM to 1 nM reducing systemic toxicities. Toxicities will be furtherreduced by encapsulating the combination therapeutic in liposomes whichhave been clinically shown to significantly reduce the in vivo toxicityof therapeutic drugs, e.g. the anti-fungal, amphotericin B.

To summarize the process of making the particle 11, of FIG. 1, inaccordance with the teaching of Castor U.S. Pat. No. 8,637,074,reference is made to FIG. 2. Supercritical, critical or near-criticalfluids with or without polar co-solvents at appropriate conditions ofpressure and temperature are utilized to solvate phospholipids,cholesterol and other nanosomal raw materials. After a specificresidence time, the resulting mixture is decompressed via a backpressureregulator (valve) though a dip tube with a nozzle into a decompressionchamber that contains phosphate-buffered saline or other biocompatiblesolution. Bubbles will form at the injection nozzle tip because of SFSdepressurization and phase-conversion into a gas, and the solvatedphospholipids will deposit out at the phase boundary of the aqueousbubble. As the bubbles detach from the nozzle into the aqueous solution,they rupture, causing bilayers of phospholipids to peel off, therebyencapsulating solute molecules and spontaneously sealing themselves toform phospholipid nanosomes. Product volatilization and oxidation aswell as processing time and organic solvent usage can be significantlyreduced with the use of supercritical, critical or near critical fluids.

A Bryoid and HDAC inhibitor will be co-encapsulated in phospholipidimmunonanosomes in the immunonanosomes apparatus shown in FIG. 2 with asupercritical, critical or near critical fluid such as carbon dioxide,nitrous oxide, fluorocarbon or alkane such as propane with or without acosolvent such ethanol. A preferred supercritical, critical or nearcritical fluid is 80% propane and 20% ethanol at 3,000 psig and 40° C.We plan to use near-critical propane which, with a dipole moment of0.084 Debyes, exhibits a much higher solvation power for phospholipidsand hydrophobic drugs. Propane is considered GRAS (generally regarded assafe) by the FDA when used under GMP conditions in the food andpharmaceutical industries. Lipid materials will be selected on the basisof previous studies and the solubility of these lipids in thesupercritical, critical or near critical fluid under appropriateoperational conditions.

The presence of cholesterol in nanosomes transforms the bilayer into anordered fluid phase over a wide temperature range, and therefore,improves the stability of nanosomes in plasma. Nanosomal compositionsare listed in Table 1.

TABLE 1 Lipid Compositions and Molar Ratios Lipid Compositions MolarRatio PC:CH 1:1 and 2:1 PC:PG:CH 1:0.1:0.4 PC:PS:CH 1:0.1:0.4DMPC:DMPG:CH 1:0.1:0.4 PC:DMPG:CH:DSPE-PEG2000 1:0.1:0.35:0.05

The supercritical, critical or near critical fluid is utilized to firstsolvate phospholipids and liposomal raw materials, then mixed with asolution of the Bryoid prior to decompression and injection into abiocompatible solution containing the HDAC inhibitor. Afterdecompression through a nozzle, the supercritical, critical or nearcritical fluid evaporates off, leaving an aqueous solution of liposomesentrapping hydrophobic Bryoid within the lipid bilayer and HDACinhibitor in the aqueous core of the phospholipid nanosomes.

Phospholipids spliced with specific antibodies are utilized to targetthe co-encapsulated drugs to the latent HIV virus. The phospholipidnanosomes are coated with antibodies or nanobodies and are referred toas immunonanosomes by using phospholipids functionalized with theligand.

One of the problems with nanosomes is phagocytosis by leukocytes and thereticuloendothelial system, which causes their rapid removal fromcirculation and makes them unavailable for uptake by tumor cells. Thisproblem is overcome by coating the particles with polyethylene glycol(PEG) which prevents them from being recognized by phagocytic cells.

PEG coating is used to produce ‘stealth’ liposomes which make themnon-recognizable by phagocytes and hence resistant to their uptake.Commercially available phospholipids with head groups linked to PEG ofvarious molecular weights will be utilized. Pegylated phospholipids willbe utilized to provide steric hindrance, increasing residence time andtherapeutic index.

We also hypothesize that targeting a combination of a Bryoid and an HDACinhibitor co-encapsulated in a long-circulation pegylatedimmunonanosomes with coatings of broadly neutralizing antibodies andanti-PD-L1 nanobodies, as shown in FIG. 1 will provide efficient HIVlatency activation and immunological depletion of latent reservoirswhile significantly reducing systemic toxicities of both Bryostatin-1and the HDAC inhibitor.

Immunonanosomes are produced by various lipid materials in the sizerange of 100 to 200 (±50) nm. Immunonanosomal suspensions of this sizerange can be filtered by a 0.22 μm filter as a final sterilization step.

Particles, such as a plurality of particle 11, are used as a suspensionin solution for administration by way of intravenous injection.

Thus, the present invention has been described in detail as an articleof manufacture and a method of treating latent viral disease with theunderstanding that those skilled in the art can modify and alter thedetailed description herein without departing from the teaching.

Therefore, the present invention should not be limited to thedescription but should encompass the subject matter of the claims thatfollow and their equivalents.

What is claimed is:
 1. A method for making a nanosome for the treatmentof latent viral diseases comprising forming an aqueous core having amixture of a hydrophilic material and an HDAC inhibitor; forming asurrounding layer having a mixture of a phospholipid and a Bryoid; andforming an outer surface encapsulating said nanosome.
 2. The method formaking a nanosome for the treatment of latent viral diseases of claim 1,further comprising: providing phospholipid material in supercritical,critical or near critical fluid; providing a Bryoid in an alcoholsolution; forming a mixture of the phospholipid fluid and the Bryoidsolution in an inline mixer; decompressing the mixture using abackpressure regulator; and injecting said mixture as a stream throughan injection nozzle into a decompression vessel containing an HDACinhibitor in a hydrophilic aqueous solution.
 3. The method for making ananosome for the treatment of latent viral diseases of claim 1, furthercomprising coating the outer surface of the nanosome with antibodieseffective in the treatment of said latent viral diseases.
 4. The methodfor making a nanosome for the treatment of latent viral diseases ofclaim 3, wherein the viruses associated with said latent viral diseasesinclude viral components, and wherein the method further comprisescoating the outer surface of the nanosome with one or more ligandsspecific for the viral components of the viruses associated with saidlatent viral diseases.
 5. The method for making a nanosome for thetreatment of latent viral diseases of claim 4, wherein the one or moreligands on the outer surface of the nanosome upregulate CD-4 cells.
 6. Amethod for making a nanosome for the treatment of latent viral diseases,said nanosome having an aqueous core having a mixture of a hydrophilicmaterial and an HDAC (histone deacetylase) inhibitor, a surroundinglayer having a mixture of a phospholipid and a Bryoid, and an outersurface; Comprising: providing phospholipid material in supercritical,critical or near critical fluid; providing a Bryoid in an alcoholsolution; forming a mixture of the phospholipid fluid and the Bryoidsolution in an inline mixer; decompressing the mixture using abackpressure regulator; and injecting said mixture as a stream throughan injection nozzle into a decompression vessel containing an HDACinhibitor in a hydrophilic aqueous solution; wherein bubbles form at theinjection nozzle, detach from the nozzle, and rupture, causing bilayersof phospholipids to peel off, encapsulating solute molecules andspontaneously sealing to form a nanosome having a core of hydrophilicmaterial and an HDAC inhibitor, a surrounding layer having aphospholipid and a Bryoid, and an outer surface encapsulating thenanosome.
 7. The method for making nanosomes for the treatment of latentviral diseases of claim 6, wherein the hydrophilic aqueous solution inwhich the HDAC inhibitor is provided further comprises alcohol.
 8. Themethod for making nanosomes for the treatment of latent viral diseasesof claim 6, wherein the alcohol solution in which the Bryoid source isprovided further comprises a buffer.
 9. The method for making a nanosomefor the treatment of latent viral diseases of claim 6, wherein theformed nanosome has a particle diameter between 100 nm and 200 nm. 10.The method for making a nanosome for the treatment of latent viraldiseases of claim 6 wherein said Bryoid is a Bryostatin.
 11. The methodfor making a nanosome for the treatment of latent viral diseases ofclaim 6, wherein said HDAC inhibitor is selected from the groupconsisting of valproic acid, Vorinostat, Romidepsin and Panobinostat.12. The method for making nanosomes the treatment of latent viraldiseases of claim 6, wherein phospholipid material is processed througha solids chamber, a mixing chamber, and a circulation loop for forming aphospholipid solution in a supercritical, critical or near criticalfluid.
 13. The method for making a nanosome for the treatment of latentviral diseases of claim 6, further comprising coating the outer surfaceof the nanosome with antibodies effective in the treatment of saidlatent viral diseases.
 14. The method for making a nanosome for thetreatment of latent viral diseases of claim 6, wherein the virusesassociated with said latent viral diseases include viral components, andwherein the method further comprises coating the outer surface of thenanosome with one or more ligands specific for the viral components ofthe viruses associated with said latent viral diseases.
 15. The methodfor making a nanosome for the treatment of latent viral diseases ofclaim 14, wherein the one or more ligands on the outer surface of thenanosome upregulate CD-4 cells.